Nucleic Acid Innovations to Manage Pathogen Sequence Divergence Foundation for Applied Molecular Evolution Steven A. Benner Zunyi Yang ABSTRACT One of the most important outcomes from synthetic biology has been the recognition that many limitations of diagnostics tools that target the DNA and RNA (collectively xNA) arise from defects in the xNA molecular framework. These defects can be fixed by changing the structure of DNA used in these assays. The Benner lab has created over a dozen reagent, enzyme, and architecture innovations based on this recognition. These are now allowing assays to move from the clinical lab to emergency rooms to physicians offices. In this progression, one problem arising from the molecular biology of the RNA viruses has remained recalci- trant. The sequences of RNA viral genomes diverge rapidly. Thus, we do not know the exact sequence of the xNA molecules that we are trying to detect in any patient. With increasing frequency, this allows RNA viruses to escape primers and probes needed to detect their xNA. This, in turn, renders simple tests unable to detect a virus with certainty. When these are intended to diagnose individual patients, this all but prevents FDA approval under anything but emergency use authorizations. Further, this uncertainty in the target xNA makes medically informative variation difficult to detect amid the medically uninformative variation. This work will introduce NextGen ?biversal? nucleic acid innovations to manage this problem. We begin with by noting that viral sequence divergence occurs under strong adaptive constraints with error biases intrinsic in relevant polymerases. Thus, transitions (purines replace purines, pyrimidines replace pyrimidines) are more common than transversions (purines replace pyrimidines, or vice versa). This means that we can manage viral genome divergence with pyrimidine biversals (Y) that pair to both A and G, and purine biversals (R) that pair to both C and T. Biversals have advantages over ?universal bases?, which create primers so promiscuous that they get lost by binding to background xNA, abundant in real biological samples. These facts set up these aims. Aim 1. Add NextGen biversals to isothermal amplification architectures, which allow diagnostics kits to move closer to points-of-sampling and points-of-care. We will benchmark their performance relative to the performance of amplifications with all-standard primers, and quantiate specificity footprints. Aim 2. Characterize the details of how polymerases handle NextGen biversals, specifically, what standard nucleotides are placed opposite these biversals by polymerases used in low-temperature amplifications. Aim 3. Integrate NextGen biversals into architectures for SNP detection that exploit ligation and cleavage. Rich preliminary data allow this project to move directly to a development stage. The emergence of Zika, Ebola, chikungunya, dengue, and other RNA viral pathogens, and the soon-to-emerge Mayoro virus, points to the immediate significance of this project. Especially innovative in this project is its integrated investigation of alternative molecular structures, the enzymes called upon to handle them, and the architectures where they will be used. These will allow FDA approvable diagnostics products to move towards points of sampling.